Chromogen for measuring the formation of hydrogen peroxide based on 1-substituted-aminoantipyrin compounds

ABSTRACT

Compounds of the general formula ##STR1## wherein R represents an alkyl group of 1 to 3 carbon atoms or the group N(R 2 ) 2 , in which R 2  represents a hydrogen atom, an alkyl group of 1 to 3 carbon atoms, or an acyl group of 1 to 3 carbon atoms, and R 1  has the same meaning as R or is a hydrogen atom, are new and are suitable as chromogens for measuring H 2  O 2  formation in enzymatic reactions.

This is a divisional application of Ser. No. 228,011, filed Jan. 23,1981, now U.S. Pat. No. 4,394,512.

The invention relates to new aminoantipyrin compounds of the generalformula ##STR2## wherein R represents an alkyl group of 1 to 3 carbonatoms or the group N(R₂)₂ in which R₂ represents a hydrogen atom, analkyl group of 1 to 3 carbon atoms or an acyl group of 1 to 3 carbonatoms, and R₁ has the same meaning as R or is a hydrogen atom, and totheir use as chromogens.

Hydrogen peroxide is the reaction product of numerous enzymaticreactions in which oxidases take part. Such reactions, such as forexample the oxidation of glycerin by glycerinoxidase or of cholesterolby cholesterinoxidase, are of great importance in analysis, and in thiscase especially for medical diagnosis.

Known methods for the determination of enzymatically formed H₂ O₂ arebased on titrimetic, potentiometric, polarographic and colorimetricmethods, and on enzymatic methods using the enzymes catalase orperoxidase. In enzymatic determinations by means of peroxidase,chromogens are used as indicators which react with hydrogen peroxide inthe presence of peroxidase with the formation of a dye which can bedetermined photometrically. One known such reagent for the H₂ O₂determination contains the Trinder indicator system (Ann. clin. Biochem.6 (1969), 24-27), in which phenol is oxidatively coupled with4-aminoantipyrin as chromogen, in the presence of peroxidase, by theaction of H₂ O₂, to form a dye which is determined photometrically.Other phenolic compounds can be used instead of phenol.

One disadvantage of 4-aminoantipyrin as chromogen in the above-describedhydrogen peroxide determination is in the poor stability of the dye thatis formed. Since in test systems it is desirable to measure against theblank value of the reagents, even an apparently slight improvement ofthe dye stability represents a great advantage with regard topracticability, for example by making it possible to provide longerreading times.

It is therefore the object of the invention to create new compoundswhich can be used as chromogens in hydrogen peroxide determinationinstead of 4-aminoantipyrin, and which have better color stabilities inthe dyes formed in the oxidative coupling with a phenolic compound.

This object is achieved by the above-described new compounds which arederivatives of 4-aminoantipyrin. In these compounds the substituents Rand R₁ can assume any position in the phenyl moiety. Compounds in whichthe moieties R and R₁ are in the para and/or ortho position arepreferred on account of their easier availability.

The compounds of the invention can be prepared by known methods, forexample by the nitration of antipyrin with two equivalents of nitricacid with the formation of dinitroantipyrin and reduction of the nitrogroups to the corresponding amino groups, for example by means of zincdust. The hydrogen atoms of the phenylic amino group can be alkylatedwith an alkylating agent, such as alkyl iodide, or acylated with anacylating agent, such as acetic acid anhydride, after masking the aminogroup in position 4 of the antipyrin, for example by forming the Schiffbase with benzaldehyde. The derivatives in accordance with theinvention, in which R or R₁ represents an alkyl group, are best preparedby setting out in a similar manner from the corresponding alkylatedphenylpyrazolones.

On the basis of the better color stability of the dyes which are formedby the compounds of the invention in the oxidative coupling with aphenolic compound and hydrogen peroxide and peroxidase, the compounds ofthe invention are especially suitable for use in methods and reagentsfor the enzymatic determination of hydrogen peroxide. In thisapplication, the common phenolic compounds can be used which can becoupled with 4-aminoantipyrin with the formation of dye. Examples ofother suitable compounds are phenol itself, other phenol derivatives,aniline derivatives, naphthol, naphthol derivatives, naphthylamine,naphthylamine derivatives, aminoquinolines, hydroxyquinolines,dihydroxyphenylacetic acid, and the like. The reaction is performed inbuffered solution. Suitable buffer substances and pH values are thesubstances and conditions known for peroxidase. pH values between 6 and9 are preferred. Otherwise, the choice of the buffer and of the pH, inthe case of a preliminary enzymatic reaction forming hydrogen peroxide,is determined mainly by the requirements with regard to buffer and thepH of the enzymes involved. These conditions are all known to the personskilled in the art and therefore require no further explanation.

A reagent for the determination of hydrogen peroxide, based onperoxidase, at least one compound of the invention, at least onephenolic compound, and buffer, can additionally contain conventionalsolvents, stabilizers and/or surface active substances. The followingquantity ratios of the essential components of this reagent have provento be especially suitable:

0.5 to 100 U/ml peroxidase,

0.05 to 20 mmol/l compound of the invention,

0.5 to 50 mmol/l phenolic compound.

Surface active agents, if they are used, are used preferably in amountsof 0.001 to 0.1 g/ml of the ready-to-use reagent solution.

The following examples will further explain the invention.

EXAMPLE 1 A. Dinitroantipyrin ##STR3##

Chemicals:

5 g (≅0.0268 mol) antipyrine

3.7 ml=3.38 g (≅2×0.0268 mol) 65% HNO₃

36 ml concentrated sulfuric acid.

Performance:

Dissolve 5 g of antipyrin in 30 ml of concentrated H₂ SO₄, add 3.7 ml ofconcentrated HNO₃ and 6 ml of H₂ SO₄ (antipyrin heats upon dissolving inH₂ SO₄ to approximately 40° C.), add HNO₃ /H₂ SO₄ drop by drop with icecooling (max. temperature +10° C.). Then heat slowly and place for 30minutes on a boiling water bath (mixture becomes increasingly darker),cool, pour onto ice (about 1 liter), remove red precipitate with suctionfilter, recrystallize from glacial acetic acid.

Yield: 4.9 g.

B. Diaminoantipyrin ##STR4##

Chemicals:

4 g dinitroantipyrin (≅0.0144 mol)

8 g zinc dust (≅0.123 mol)

Performance:

Dissolve 4 g of dinitroantipyrin in 50 ml of concentrated hydrochloricacid with cooling (max. 20° C.), add zinc dust (about 8 g) untilsolution is colorless. Add 40% NaOH/NaOH tablets to pH 7; thickprecipitate forms which is difficult to suction filter (not much morecan be extracted with CHCl₃). Adjust mother liquor to pH 11.5 withCHCl₃, extract, concentrate. Sample dissolves in water, gives a dark redcolor with DCP/FeCl₃.

Yield: 0.4 g.

EXAMPLE 2 A. Preparation of the 4-benzal compound of1-(p-aminophenyl)-2,3-dimethyl-4-aminopyrazolone-5 (II) ##STR5##

1.09 g (5 mmol) of diaminoantipyrin (I) is dissolved in 15 ml of waterand adjusted to pH 10 with 1N soda lye. 0.53 ml (5 mmol) of benzaldehydeis added. The mixture is stirred vigorously for 20 hours at roomtemperature. Then the crystalline product is removed with a suctionfilter and washed with water and ether.

Yield: 82%.

The substance II which is obtained is used without further refiningoperations for the following reactions.

B. 4-Benzal compound of1-(p-diethylamino-phenyl)-2,3-dimethyl-4-amino-pyrazolone-5

9.18 g of the 4-benzal compound of diaminoantipyrin (0.03 mol) isdissolved in 250 ml of ethylene glycol dimethyl ether with refluxing.Then the mixture is cooled to 60° C. Then 56 g of potassium carbonate aswell as 14.6 ml=28.0 g of ethyl iodide (0.18 mol) is added. The mixtureis then refluxed for 9 hours and the potassium carbonate is removed witha suction filter. The filtrate is vacuum-dried. Residue: 12.16 g. Theresidue is chromatographed (ethanol) through a silica gel column. Thechromatographically purest fractions are crystallized from diethylether.

Yield: 3.1 g.

DC Results: Uniform [=chromatography pure].

C. 1-(p-Diethylamino-phenyl)-2,3-dimethyl-4-amino-pyrazolone-5 ##STR6##

1.0 g of the product of step 2 is dissolved in 35 ml of hydrochloricacid (2N) at room temperature and then stirred for 2.5 hours. The BVcleavage is traced by chromatography. The mixture is extracted threetimes with 30 ml of chloroform each time to separate the benzaldehyde.Then the acid, aqueous phase is adjusted with caustic soda solution(33%) to pH 10.0 and again extracted with chloroform (thrice with 30 mleach time). The chloroform is withdrawn in vacuo. Residue: 0.855 g. Theresidue is subjected to another silica gel treatment (chloroform/ethanol1:1).

The yield of chromatographically uniform substance is 0.356 g. MS: 274.

EXAMPLE 3 A. 4-Benzal compound of1-(p-acetamino-phenyl)-2,3-dimethyl-4-aminopyrazolone-5

15.3 g of benzal compound of diaminoantipyrin (1/20 mol) is dissolved in60 ml of dimethylformamide at 45° to 50° C. The addition of 10.2 g ofacetic anhydride is performed at room temperature, whereupon thetemperature increases to 50° and spontaneous crystallization takesplace. After two hours the crystallizate is separated with a suctionfilter and washed with DMF. The substance is chromatographically pure.

Yield: 14.6 g. M.P. 279° to 282° C. MS: 348.

B. 1-(p-Acetamino-phenyl)-2,3-dimethyl-4-aminopyrazolone-5 ##STR7##

10.0 g of the product of step A is dissolved in 350 ml of hydrochloricacid (2N) at room temperature and then cooled to 10° C., whereupon thecleavage of the benzal compound takes place, which is complete withinone hour. The acid solution is extracted with chloroform to remove thebenzaldehyde. Then the pH is adjusted to 10.0 to 11.0 with soda lye atroom temperature, and the alkaline solution is saturated with NaCl. Thenfollows another extraction with chloroform. The chloroform is withdrawnin vacuo. Residue: 6.1 g. This is recrystallized from 36 ml of ethanol.

Yield: 3.6 g. M.P. 210° C. (Z). MS: 260. Chromatographically pure.

EXAMPLE 4 1-(Amino-p-tolyl)-2,3-dimethyl-4-amino-pyrazolone-5

The synthesis is performed in accordance with the following scheme:##STR8##

A. 1-(p-Tolyl)-2,3-dimethyl-pyrazolone-5

18.8 g of 1-(p-tolyl)-3-methyl-pyrazolone-5 is partially melted at 110°to 115° C., and 10.5 ml of dimethyl sulfate (0.11 mol) is added drop bydrop over a period of 30 minutes. Then the mixture is heated at 170° C.for 5 hours. After cooling the mixture below 100° C., 35 ml of water isadded. Then the mixture is refluxed for another 5 hours. After themixture is cooled to 30° C., 25 ml of soda lye (33%) is added. pH=10.0to 11.0.

The mixture is again stirred for 5 hours at 90° to 95° C. Then it iscooled to room temperature and extracted four times with 100 ml ofchloroform each time. The chloroform is withdrawn by distillation invacuo.

Residue: 19.9 g. M.P. 132°-134° C. MS: 202. DC Results: Uniform[=chromatrographically pure.]

B. Dinitro compound of 1-(p-tolyl)-2,3-dimethylpyrazolone-5

10.73 g of the product of step A is added at 25° C. to 60 ml ofconcentrated sulfuric acid. Then a nitrating mixture of 8.05 ml ofnitric acid (65%) and 12 ml of concentrated sulfuric acid is added dropby drop over a period of two hours with ice cooling. The temperature isto be from 5° to 7° C. After that the mixture is stirred for 30 minutesat room temperature and heated for an additional 30 minutes at 100° C.

After the solution has cooled to room temperature it is poured onto 0.5kg of ice. After one more hour the crystallizate is separated with asuction filter and recrystallized from 280 ml of glacial acetic acid.

Yield: 11.5 g. M.P. 274°-275° C. MS: 292.

C. 1-Amino-p-tolyl)-2,3-dimethyl-4-aminopyrazolone-5

10.3 g of the product of step B is dissolved in 95 ml of concentratedhydrochloric acid at room temperature. 30 g of zinc dust is added, withstirring, at between 5° and 25° C. (icewater cooling), over a period of1.5 hours until the solution is decolorized. Then 20 g of soda plus atotal of 45 g of caustic soda flakes is added. The pH is to be higherthan 10.0.

The thick mixture is dried at 40° C. in the vacuum dryer. The dryresidue is pulverized and then extracted with chloroform for threehours. Residue: 7.47 g. Recrystallization is performed from 27.5 ml ofethanol.

Yield: 4.0 g. M.P. 165°-169° C. MS: 232. DC Results: Uniform[=chromatographically pure.]

EXAMPLE 5 1-(o-Ethyl-amino-phenyl)-2,3-dimethyl-4-amino-pyrazolone-5

The synthesis is performed in accordance with the following reactionprogram: ##STR9##

A. 1-(o-Ethyl-phenyl)-2,3-dimethyl-pyrazolone-5

The methylation in the second position is performed as described inExample 4A.

Input:

20.2 g of 1-(o-ethyl-phenyl)-3-methyl-pyrazolone-5 (0.1 mol), 10.5 mldimethylsulfate (0.11 mol). The substance becomes solid upon heating at170° C. (5 hours). The amount of water required for dissolution is 60ml. Residue after chloroform extraction: 20.4 g. Purification isperformed on a silica gel column.

Yield: 16.7 g. M.P. 61°-61.5° C. MS: 216. Chromatographically pure.

B. Dinitro compound of 1-(o-ethyl-phenyl)-2,3-dimethylpyrazolone-5

The nitration was performed as described in Example 4B.

Input:

11.47 g 1-(o-ethyl-phenyl)-2,3-dimethyl-pyrazolone-5

60 ml sulfuric acid (conc.)

    ______________________________________                                        8.05 ml nitric acid (65%)                                                                       nitrating mixture                                           12 ml sulfuric acid (conc.)                                                   ______________________________________                                    

Residue: 14.7 g

The recrystallization was performed from glacial acetic acid (390 ml).

Yield: 11.07 g. M.P. 269°-272° C. MS: 306.

C. 1-(o-Ethylaminophenyl)-2,3-dimethyl-4-amino-pyrazolone-5

The reduction of the dinitro compound was performed as described inExample 4C.

Input:

10.8 g dinitro compound,

235 ml hydrochloric acid

36.5 g zinc dust

20 g soda

100 g caustic soda flakes

Chloroform residue: 7.7 g

Reprecipitation from chloroform/diisopropyl ether. For this purpose 6.0g of the residue is dissolved in 140 ml of chloroform and thenprecipitated with 200 ml of diisopropyl ether.

Yield: 5.3 g. M.P. 179°-181° C. MS: 246. DC Results: Uniform[=chromatographically pure.]

EXAMPLE 6

Determination of hydrogen peroxide formation in the oxidation ofglycerin by glycerinoxidase.

Two reagents are prepared:

    ______________________________________                                        Reagent 1:                                                                              0.1 mol/l triethanolamine/HCl buffer, pH 8.0                                  3.6 mmol/l or 2 g/l isotridecyl ether                                         4.7 mmol/l or 2 g/l sodium cholate                                            10 mmol/l p-chlorophenol                                                      0.5 mmol/l amino-substituted 4-aminoantipyrin                                 10 U/ml peroxidase                                                  Reagent 2:                                                                              500 U/ml glycerinoxidase                                            ______________________________________                                    

For the performance of the determination, 2 ml of reagent 1 and 0.2 mlof reagent 2 were pipetted into a colorimeter cell. The extinction E₁ isread on the light meter at 546 nm. Then the reaction is started by theaddition of 20 microliters of sample. After 20 minutes of reaction, theextinction E₂ is read. The incubation temperature is 25° C.

The evaluation is performed on the basis of a straight calibration lineat which the measured extinction difference ΔE=E₂ -E₁ of a standardsolution of glycerin is related to the glycerin concentration.

The concentration in the test solution are:

0.09 mol/l triethanolamine/HCl buffer, pH 8.0

1.8 g/l (3.2 mmol/l) isotridecyl ether

4.3 mmol/l sodium cholate

9 mmol/l p-chlorophenol

0.45 mmol/l aminosubstituted 4-aminoantipyrine

9 U/ml peroxidase 45 U/ml glycerinoxidase.

EXAMPLE 7

The color stability of five compounds in accordance with the inventionwas tested. 4-Aminoantipyrin and sulfonated 4-aminoantipyrin were usedas compounds for comparison.

With these standard compounds and the new compounds, the extinction ε,the stability of the dye formed and λ_(max) were determined, usingp-chlorophenol, 2,4-dichlorophenol and ethylhydroxytoluidine(sulfonated) as phenolic couplers. The test system consisted of H₂ O₂/POD/dye components.

Performance of the test:

The following reagents were used:

    ______________________________________                                        Reagent 1: 0.1 mol/l potassium phosphate buffer, pH 8.0                                  0.1 mmol/l 4-aminoantipyrin derivative                                        1.0 mmol/l phenol component                                                   2 U/ml POD                                                         Reagent 2: 0.01 mol/l hydrogen peroxide solution.                             ______________________________________                                    

Sample for determination:

Measuring radiation: Hg 546 nm; depth of substance in the cell: 1 cm;incubation temperature: room temperature.

2.0 ml of reagent 1 is pipetted into the cell, 10 microliters of reagent2 (sample) are added and mixed. The extinction is traced for 70 minutes.A blank value is determined for each test, adding buffer instead of thehydrogen peroxide solution to reagent 1.

The results are summarized in the table below. The stated percentage ofcolor stability (60 minutes at room temperature) relates to thedifference which is produced by hydrogen peroxide with respect to themeasuring signal. From the table it can be seen that λ_(max) isnegligibly affected by the new compounds of the invention, as is alsothe ε value. The new compounds of the invention are decidedly superiorin color stability to the standards used for comparison.

                                      TABLE                                       __________________________________________________________________________              Compounds Used                                                                for Comparison                                                                          Compounds Used in Accordance with the invention                          4-amino-  1-(o-ethyl-                                                                           1-(p-acet-                                                                            1-(amino-p-                                                                         1-(p-diethyl-                                 anti-                                                                              4-amino-                                                                           aminophenyl)-                                                                         aminophenyl)-                                                                         tolyl)-2,3-                                                                         aminophenyl)-                            4-amino-                                                                           pyrin                                                                              anti-                                                                              2,3-dimethyl-                                                                         2,3-dimethyl-                                                                         dimethyl-4-                                                                         2,3-dimethyl-                            anti-                                                                              (sulfon-                                                                           amine                                                                              4-aminopyra-                                                                          4-aminopyra-                                                                          aminopyra-                                                                          4-aminopyra-                   Phenolic coupler                                                                        pyrin                                                                              ated)                                                                              pyrin                                                                              zolone-(5)                                                                            zolone-(5)                                                                            zolone-(5)                                                                          zolone-(5)                     __________________________________________________________________________    p-chlorophenol                                                                (cm.sup.2 /μmol)                                                                     12   12   14   9       19      20    19                             color stability                                                                         +5%  +4%  +1%  +0%     +0%     +0%   +1%                            max       500  502  505  503     502     503                                  2,4-dichlorophenol                                                            (cm.sup.2 /μmol)                                                                     10   18   27   25      22      15    41                             color stability                                                                         +2%  +7%  +1%  -1%     +0%     +2%   +1%                            max       505       507  508     507     508   502                            Ethylhydroxy-                                                                 toluidine (sul-                                                               fonated)                                                                      (cm.sup.2 /μmol)                                                                     33   32   36   23      25      25    37                             color stability                                                                         +16% -11% -2%  -3%     -7%     -5%   -4%                            max       550  546  550  438     546     550   546                            __________________________________________________________________________

I claim:
 1. In a method for the determination of enzymatically formedhydrogen peroxide of the type wherein phenol or other phenolic compoundis oxidatively coupled with 4-aminoantipyrine as the chromogen, in thepresence of peroxidase, by the action of hydrogen peroxide, to form adye which is determined photometrically, the improvement comprisingreplacing the 4-aminoantipyrine with a compound of the formula ##STR10##wherein R represents the group N(R₂)₂, in which R₂ represents an alkylgroup of 1 to 3 carbon atoms or one R₂ is an acyl group of 1 to 3 carbonatoms and the other R₂ is hydrogen or an alkyl group of 1 to 3 carbonatoms, and R₁ has the same meaning as R or is a hydrogen atom or NH₂. 2.The method of claim 1 wherein R is NH-acetyl.
 3. The method of claim 1wherein R is N(R₂)₂ in which R₂ is an alkyl group of 1 to 3 carbonatoms.
 4. The method of claim 1 wherein R₁ is hydrogen.
 5. The method ofclaim 1 wherein R₁ is NH₂.